JP4366507B2 - Method for quantifying oxidized protein - Google Patents

Description

  The present invention relates to a method for quantifying an oxidized protein capable of highly sensitively quantifying a carbonylated protein that has undergone oxidative denaturation, and further, a labeled reagent for quantifying an oxidized protein and an oxidized protein used for quantifying the oxidized protein. The present invention relates to a quantitative labeling reagent kit.

  Protein oxidation is presumed to be related to diseases such as arteriosclerosis, rheumatoid arthritis, emphysema, neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, etc.), aging, acute pancreatitis, cancer, etc. There is a demand for highly sensitive quantitative determination. For example, there is a theory that oxidative degeneration of low density lipoprotein (LDL) having a role of transporting cholesterol triggers arteriosclerosis, and elucidation of active species causing LDL oxidation is awaited. In order to elucidate, it is necessary to elucidate the oxidation site of LDL and further information from the product side.

  As protein quantification techniques, spectroscopic analysis has been the mainstream, and various quantification methods have been studied. For example, 2,4-dinitrophenylhydrazine (DNPH) forms a stable Schiff base with a carbonylated protein that has undergone oxidative modification, and thus quantifies the carbonylated protein by measuring the absorption of ultraviolet light. (See, for example, Non-Patent Document 1). In addition, two-dimensional electrophoresis is a method in which proteins are developed with different isoelectric points and molecular weights. After staining with Coomassie Brilliant Blue dye, etc., each spot area, color intensity, etc. are quantified and quantified ( For example, refer nonpatent literature 2.).

  As described above, DNPH has been used as a reagent for detecting carbonylation of proteins due to oxidative stress, but since the detection method was absorption of ultraviolet light or antibody detection using an anti-DNP antibody, There was no discussion about the site. Furthermore, since the detection method is spectral analysis of the ultraviolet wavelength light, the detection sensitivity is limited.

  On the other hand, two-dimensional electrophoresis has a problem with linearity, and for example, the size of a spot often does not reflect the actual amount. In addition, since image analysis is a three-dimensional evaluation method that takes into account the thickness of the electrophoresis gel, it has a problem that it requires much labor and time.

  Under such circumstances, a new protein analysis method using mass spectrometry has been proposed (see, for example, Patent Document 1 and Patent Document 2). For example, Patent Document 1 discloses a step of preparing a sample including a plurality of specimens labeled with isotope reagents having different molecular weights, a step of separating the sample by chromatography, and a tandem type capable of performing multistage dissociation measurement of the separated sample. Disclosed is a mass spectrometry method comprising a step of performing mass analysis by a mass spectrometer, a step of analyzing a mass spectrometry result in real time, and a step of utilizing an analysis result of at least one sample in mass analysis of another sample in real time. ing. In the invention described in Patent Document 1, proteins, peptides obtained by degrading proteins, and the like are cited as specimens, and biopolymers can be identified and quantified with high accuracy and high throughput.

The invention described in Patent Document 2 relates to a tag for oxidative damage protein analysis used for detection, quantification, and determination of an oxidative damage site in vivo, and a method for analyzing oxidative damage protein using the same It is. In the method for analyzing oxidative damage protein disclosed in Patent Document 2, the analysis tag and the isotope-labeled analysis tag are mixed with a sample, and the mixture retains a metal atom that binds to the affinity peptide tag of the analysis tag. The complex of the oxidatively damaged protein and the tag for analysis is recovered from this carrier, and the tablet peak due to the difference in molecular weight is detected by mass spectrometry. The tag for oxidative damage protein analysis comprises at least one residue having an affinity peptide, a linker and a reactive group, has at least 3 (X-His) units, and X is any amino acid or amino acid derivative. is there.
Methods Enzymol., 186: 464-478 (1990) J. Biol. Chem. 250: 4007-4021 (1975) JP 2005-345332 A JP 2005-315688 A

  The invention described in Patent Document 1 and the invention described in Patent Document 2 both identify proteins to be analyzed using a pair of peaks (doublet spectrum) expressed by mass spectrometry by labeling with an isotope reagent. This has the advantage that identification is easier and more reliable than when labeling is simply performed with a labeling reagent.

  However, in protein mass spectrometry, various peaks are expressed, and simply labeling with an isotope reagent cannot rule out the possibility that any pair of peaks overlaps with other peaks, and can be easily and reliably identified. Not necessarily.

  Regarding the invention described in Patent Document 1, no reference is made to the analysis of carbonylated protein that has undergone oxidative modification, and no specific description of, for example, the labeled isotope reagent is found. On the other hand, in the invention described in Patent Document 2, a linker or the like is required for the analysis tag, and not only the synthesis of the analysis tag is complicated, but also the analysis accuracy may decrease due to an increase in fragment ions. is there. Further, in the invention described in Patent Document 2, the operation is complicated because it is necessary to make contact with a carrier that holds a metal atom that binds to the affinity peptide tag of the analysis tag.

  The present invention has been proposed in view of such problems of the prior art, and it is possible to easily and reliably identify a peak derived from an oxidized protein labeled in mass spectrometry. It is an object of the present invention to provide a method for quantifying oxidized protein capable of rapid quantification. The present invention also provides an oxidized protein quantification labeling reagent and an oxidized protein quantification labeling reagent kit that are easy to synthesize and that can specifically bind to and label a specific site of an oxidized protein (carbonylated protein). The purpose is to provide.

The method for quantifying an oxidized protein of the present invention is a method for quantifying an oxidized protein in which oxidized protein that has undergone oxidative modification is labeled with a labeling reagent and quantified by mass spectrometry, wherein the labeled reagent reacts with the oxidized protein. Using the first labeling reagent and the second labeling reagent having the same chemical structure as the first labeling reagent and having at least some of the constituent atoms substituted with isotopes of the atoms, A peak derived from the oxidized protein is obtained by mixing the oxidized protein labeled with the oxidized protein labeled with the second labeling reagent, changing the mixing ratio, and performing the mass spectrometry at each mixing ratio. It is characterized by specifying .

  When the oxidized protein labeled with the first labeling reagent and the oxidized protein labeled with the second labeling reagent are mixed and subjected to mass spectrometry, a mass difference corresponding to the mass difference between the first labeling reagent and the second labeling reagent is obtained. Peak pairs appear in the spectrum. However, this alone may cause the paired peaks to overlap with other peaks, and it is difficult to reliably determine them. Therefore, in the present invention, the mass spectrometry is performed by changing the ratio of the oxidized protein labeled with the first labeling reagent and the oxidized protein labeled with the second labeling reagent. For example, when mass spectrometry is performed with the ratio of the oxidized protein labeled with the first labeling reagent larger than the ratio of the oxidized protein labeled with the second labeling reagent, and the ratio of the oxidized protein labeled with the first labeling reagent Is smaller than the ratio of the oxidized protein labeled with the second labeling reagent, the relationship between the peak heights of the pair of peaks is reversed. The peak derived from the labeled oxidized protein is identified and quantified from the reversal of the height of the paired peak.

  On the other hand, the labeled reagent for quantifying oxidized protein of the present invention is characterized by containing 2,4-dinitrophenylhydrazine in which 6 carbon atoms of the phenyl group are substituted with carbon isotopes. Moreover, the labeled reagent kit for quantifying oxidized protein of the present invention includes a first title reagent containing 2,4-dinitrophenylhydrazine, and 2,4-diphenyl having 6 carbon atoms substituted with carbon isotopes. And a second labeling reagent containing dinitrophenylhydrazine.

2,4-dinitrophenylhydrazine (DNPH) forms a stable Schiff base with an oxidized protein (carbonylated protein) that has undergone oxidative modification due to oxidative stress or the like, and binds to and labels the carbonylated protein. When the carbon of the phenyl group of 2,4-dinitrophenylhydrazine is replaced with a carbon isotope ( ¹³ C), it becomes 2,4-dinitrophenylhydrazine ( ¹³ C ₆ -DNPH) which differs only in molecular weight by 6 and thereby carbonylation. When the protein is labeled, it is separated as the same peak as when labeled with DNPH in chromatography, for example. Thus, when a carbonylated protein is labeled with the DNPH and ¹³ C-DNPH, separated by chromatography and subjected to mass spectrometry, a peak pair having a mass difference of 6 appears in the spectrum. Since the labeling reagent for protein quantification ( ¹³ C ₆ -DNPH) of the present invention does not require a linker or the like, it is easy to synthesize and does not lower the analysis accuracy.

  According to the present invention, an oxidized protein (carbonylated protein) can be quantified with high sensitivity and speed, and for example, useful information can be provided for analyzing a causal relationship between protein oxidation and a disease. Expected. Further, according to the present invention, it is possible to quantify oxidized protein without requiring time and labor, and it is possible to quantify oxidized protein with good reproducibility without requiring skill.

  Hereinafter, a method for quantifying oxidized protein, a labeled reagent for quantifying oxidized protein, and a labeled reagent kit for quantifying oxidized protein will be described in detail.

  In the method for quantifying oxidized protein of the present invention, oxidized protein (for example, carbonylated protein) subjected to oxidative modification is labeled with a labeling reagent, and mass spectrometry is performed. Proteins undergo oxidative denaturation due to oxidative stress and the like, and some of the amino acids are oxidized to produce various oxidation products. Table 1 shows the main oxidation products of amino acids and their mass changes.

  When the protein undergoes oxidative denaturation, as shown in Table 1, the mass changes according to the oxidation product of amino acids. For example, if the amino acid is carbonylated, the mass change is +14. Therefore, when mass spectrometry is performed, as shown in FIG. 1, a peak B derived from an oxidized protein (carbonyl protein) appears at a position of +14 with respect to a peak A derived from a target protein.

  As shown in Table 1, various functional groups (for example, carbonyl groups) are formed in the oxidized protein by oxidation. When a labeling reagent that reacts with the functional group is reacted, the labeled reagent is bound to form a labeled protein. The mass difference (molecular mass difference) M between the labeled protein and the unlabeled oxidized protein is obtained by subtracting the molecular mass of the molecule lost by the reaction from the molecular mass of the labeling reagent, as shown in FIG. The peak C derived from the labeled protein appears at the position of + M with respect to the peak B derived from the oxidized protein.

Here, in the present invention, as the labeling reagent, the first labeling reagent that reacts with the oxidized protein, the chemical structure identical to that of the first labeling reagent, and at least a part of the constituent atoms is an isotope of the atom Labeling is carried out using the second labeling reagent substituted with. The second labeling reagent has the same chemical structure as the first labeling reagent, and at least a part of the constituent atoms is substituted with an isotope of the atom. Therefore, the substituted isotope The molecular mass differs from that of the first labeling reagent by the amount. For example, if 6 carbon atoms of the phenyl group of the second labeling reagent are substituted with ¹³ C, which is a stable isotope, the mass difference between the first labeling reagent and the second labeling reagent is 6. . When the oxidized protein is labeled using the first labeling reagent and the second labeling reagent, the first labeled protein labeled with the first labeling reagent and the second labeled with the second labeling reagent A labeled protein is generated, and a peak C derived from the first labeled protein and a peak D derived from the second labeled protein and derived from the peak C appear on the mass spectrometry chart. The mass difference between these peaks C and D is 6.

  As described above, by using the first labeling reagent and the second labeling reagent, peak pairs (peak C and peak D) appear, and by confirming these peak pairs, the peak derived from the oxidized protein is confirmed. can do. However, in protein mass spectrometry, a very large number of peaks often appear, and the peak pair is buried in these peaks and may not be correctly identified.

  Therefore, in the present invention, the labeling is performed by changing the ratio of the first labeling reagent and the second labeling reagent, and mass spectrometry is performed in each case, so that the peak pair can be reliably confirmed. Specifically, first, labeling is performed with a small ratio of the first labeling reagent and a large ratio of the second labeling reagent, and mass spectrometry is performed. Then, as shown to Fig.2 (a), it originates in the 2nd labeled protein labeled with the 2nd labeling reagent rather than the peak C derived from the 1st labeled protein labeled with the 1st labeling reagent. Peak D has a higher height (peak intensity). Next, first, labeling is performed with the ratio of the first labeling reagent being large and the ratio of the second labeling reagent being small, and mass spectrometry is performed. Then, as shown in FIG. 2 (b), it is derived from the second labeled protein labeled with the second labeling reagent rather than the peak C derived from the first labeled protein labeled with the first labeling reagent. Peak D has a lower height (peak intensity).

  Therefore, if the labeling and mass spectrometry are performed twice, a peak pair whose height is inverted can be identified as a peak derived from an oxidized protein. If the reversal of the peak height is recognized, it is possible to reliably determine the target peak pair even if there are many peaks. As a method for performing the labeling by changing the ratio of the first labeling reagent and the second labeling reagent, for example, after labeling the oxidized protein with the first labeling reagent and the second labeling reagent, these labels are used. What is necessary is just to mix the oxidized protein which changed the ratio. Although the case where the labeling and the mass spectrometry are performed twice is described here, the labeling and the mass spectrometry in which the ratio between the first labeling reagent and the second labeling reagent is changed can be performed three times or more.

  The mass spectrometry can be performed without any pretreatment on the labeled analysis sample (labeled protein). When the molecular weight of a protein is very large, for example, enzymatic digestion can be performed after decomposition into a component having a small molecular weight to some extent. A peak pair may be observed for a plurality of components by the decomposition, and the peak pair may be confirmed more clearly.

  Moreover, in the said mass spectrometry, it is also possible to perform the tandem mass spectrometry (MS / MS measurement) about the peak derived from an oxidation protein, and to analyze the oxidation site | part of an oxidation protein. In the MS / MS measurement, all ions generated in the ion source are separated by a first mass spectrometer, and only a target peak is selectively fragmented and analyzed by a second mass spectrometer. In MS / MS measurement of peptides, analysis of amino acid sequences is possible.

  Next, the labeling reagent used in the above-described method for quantifying oxidized protein will be described. As described above, in the method for quantifying oxidized protein of the present invention, a labeling reagent for labeling the oxidized protein is required. Here, the labeling reagent needs to react with the oxidized site of the oxidized protein, and should be selected according to the oxidation product of the amino acid shown in Table 1. In the present invention, a carbonylated protein that has undergone oxidative denaturation and is carbonylated is mainly the subject of measurement, and 2,4-dinitrophenylhydrazine (DNPH) shown in Chemical Formula 1 is used as a labeling reagent.

As shown in FIG. 3A, the DNPH reacts with a carbonyl group of an oxidized protein (carbonylated protein) to form a stable Schiff base to produce a DNPH protein. The difference in mass number between the DNPH protein and the carbonylated protein is the mass number of DNPH (198) −the mass number of H ₂ O (18) = 180, and the mass between the DNPH protein and the protein before being carbonylated. The number difference is 194. While using the DNPH as a first labeling reagent, 2,4-dinitrophenylhydrazine ( ¹³ C-DNPH) in which 6 carbon atoms of the phenyl group of DNPH are replaced with a stable isotope ( ¹³ C) is used as the second labeling reagent. Used as a labeling reagent.

The ¹³ C-DNPH, as shown in FIG. 3 (b), similarly to the DNPH, reacts with the carbonyl group of the carbonylation ^protein, to generate a 13 C-DNPH of protein. ¹³ C-DNPH has a mass number 6 larger than that of DNPH, and the mass number difference between the ¹³ C-DNPH protein and the DNPH protein is 6. Therefore, when the carbonylated protein labeled with DNPH and the carbonylated protein labeled with ¹³ C-DNPH are mixed and subjected to mass spectrometry, a peak pair with a mass difference of 6 is observed. In general, when a carbonylated protein is identified by mass spectrometry, it is divided by a proteolytic enzyme such as trypsin to make a small peptide and then measured.

The ¹³ C-DNPH was synthesized for the first time by the present inventors, and synthesized according to a conventional method using benzene in which 6 carbon atoms are substituted with a stable isotope ( ¹³ C) as a starting material. Can do. FIG. 4 shows a synthesis scheme of ¹³ C-DNPH. To synthesize ¹³ C-DNPH, benzene is brominated to form bromobenzene, and a nitro group is introduced into this to form 2,4-dinitrobromobenzene. Hydrazine hydrate is allowed to act on the synthesized 2,4-dinitrobromobenzene to obtain 2,4-dinitrophenylhydrazine. By using benzene in which 6 carbon atoms are substituted with a stable isotope ( ¹³ C) as the starting material, 6 carbon atoms of the phenyl group are substituted with stable isotopes ( ¹³ C) 2,4 -Dinitrophenylhydrazine ( ¹³ C-DNPH) can be synthesized.

  Hereinafter, specific examples to which the present invention is applied will be described based on experimental results.

Synthesis of ¹³ C ₆ -DNPH To 0.441 g (5.65 mmol) of benzene ( ¹³ C ₆ ) in which 6 carbon atoms are substituted with a stable isotope ( ¹³ C), 0.1 mg of Fe and 0.137 ml of Br ₂ (5.29 m) Mol) was added and stirred at 55 ° C. for 15 minutes. This was cooled to room temperature, 10% aqueous sodium hydroxide solution was added, and the mixture was extracted with diethyl ether. After washing with water, distillation was performed to obtain bromobenzene.

Next, 7.5 ml of sulfuric acid (H ₂ SO ₄ ) and 5.0 ml of nitric acid (HNO ₃ ) were mixed, heated to 85 ° C. with stirring, and the bromobenzene synthesized earlier was added. Further, stirring was continued at 85 ° C., and then cooled to room temperature. After cooling, the mixture was ice-cooled and extracted with diethyl ether. After washing with water, the solution was concentrated under reduced pressure (evaporated) and developed by thin layer chromatography to determine the separation conditions for the target compound. This was purified through a silica gel column, and the resulting product was analyzed by ¹ H-NMR. As a result, formation of dinitrobromobenzene was confirmed. The yield of dinitrobromobenzene was 316.3 mg (1.25 mmol), and the total yield from benzene ( ¹³ C ₆ ) was 22.1%.

Subsequently, 4.0 ml of ethanol was added to the dinitrobromobenzene, and 0.40 ml (14.4 mmol) of hydrazine hydrate and 1.50 ml of ethanol were added with stirring at 65 ° C. Stirring was continued at 65 ° C., and then cooled to room temperature and filtered. This was washed with cooled ethanol and dried under reduced pressure to obtain ¹³ C ₆ -DNPH. The yield of ¹³ C ₆ -DNPH obtained was 201.0 mg (0.99 mmol), and the yield in this step was 78.8%. The total yield from the raw material benzene ( ¹³ C ₆ ) was 17.4%.

Labeling and mass spectrometry of oxidized myoglobin ( labeling with ¹² C-DNPH)
100 mM PBS (pH 7.4) and 2 nL of NaOCl (diluted to 1/100) were added to 100 nL (10 nmol) of 0.1 mM protein (myoglobin) and reacted at -85 ° C for 30 minutes. As a result, the protein was oxidized (carbonylated).

Next, the reaction product was precipitated with acetone, the following reagents were sequentially added, and the mixture was reacted at room temperature for 30 minutes in a dark room. Thus, the carbonylated myoglobin was converted to ¹² C-DNPH.
10 mM Tris-HCl (pH 8.0): 50 μL
10% SDS: 5 μL
10 mM ¹² C-DNPH / 2N-HCl: 50 μL

(Labeling with ¹³ C-DNPH)
100 mM PBS (pH 7.4) and 2 nL of NaOCl (diluted to 1/100) were added to 30 nL (3 nmol) of 0.1 mM protein (myoglobin), and reacted at -85 ° C for 30 minutes. As a result, the protein was oxidized (carbonylated).

Next, the reaction product was precipitated with acetone, the following reagents were sequentially added, and the mixture was reacted at room temperature for 30 minutes in a dark room. Thus, the carbonylated myoglobin was converted to ¹³ C-DNPH.
10 mM Tris-HCl (pH 8.0): 50 μL
10% SDS: 5 μL
10 mM ¹³ C-DNPH / 2N-HCl: 50 μL

(Mixing and mass spectrometry of ¹² C-DNPH protein and ¹³ C-DNPH protein)
The synthesized ¹² C-DNPH protein and ¹³ C-DNPH protein were mixed ( ¹² C-DNPH protein: ¹³ C-DNPH protein = 100: 30), precipitated with acetone, and then 6M urea / 25 mM. 50 μL of ammonium hydrogen carbonate and 2 μL of 200 mM DTT were added and reacted at 37 ° C. for 1 hour. Subsequently, 450 μL of 25 mM ammonium bicarbonate and trypsin were added, and enzyme digestion was performed at 37 ° C. for a whole day and night. After concentration under reduced pressure to 10 μL to 20 μL, 0.5 μL was subjected to mass spectrometry using ZipTipC18 (eluted with 10 μL). CHCA / 50% acetonitrile was used as the matrix solvent. Mass spectrometry was performed using MALDI-TOF / MS (matrix-assisted laser desorption / ionization time-of-flight mass spectrometer).

(Results of mass spectrometry)
FIG. 5 is a mass spectrometry chart by MALDI-TOF / MS. In FIG. 5, two pairs of peaks, a peak with mass 786.461 and a peak with mass 792.475, and a peak with mass 808.439 and a peak with mass 8144.489 are observed as a peak pair with a mass number difference of 6. It was. In any peak pair, the peak derived from the ¹² C-DNPH protein (the peak with mass 786.461 and the peak with mass 808.439) has a higher peak intensity.

Thus, next, ¹² C-DNPH protein: ¹³ C-DNPH protein = 30: 100 (molar ratio) was adjusted, and the others were similarly labeled with ¹² C-DNPH and labeled with ¹³ C-DNPH. , ¹² C-DNPH protein and ¹³ C-DNPH protein were mixed and mass analyzed. FIG. 6 shows a mass spectrometry chart in this case.

Also in this case, two pairs of peaks, a mass of 786.461 and a mass of 792.475, and a mass of 808.439 and a mass of 8144.489, were observed as a peak pair with a mass number difference of 6. It was. However, in any peak pair, the peak derived from ¹² C-DNPH protein (the peak with mass 786.461 and the peak with mass 808.439) has a smaller peak intensity.

  From these facts, it was possible to determine that the two pairs of peaks were derived from myoglobin that had undergone oxidative modification. Moreover, the oxidation site | part of myoglobin was able to be analyzed by performing MS / MS measurement about these peaks.

Labeling with oxidized lysozyme and mass spectrometry Oxidation was performed in the same manner as myoglobin, and labeling with ¹² C-DNPH and labeling with ¹³ C-DNPH were performed. Furthermore, these were mixed and mass spectrometry was performed. The oxidation method, labeling method, mixing, and mass spectrometry were performed according to the case of myoglobin.

FIG. 7 is a mass spectrometry chart when ¹² C-DNPH protein: ¹³ C-DNPH protein = 100: 30, and FIG. 8 is ¹² C-DNPH protein: ¹³ C-DNPH protein = 30: 100 (mole). Ratio) is a mass spectrometry chart. Three pairs of peaks, a mass 785.436 peak and a mass 791.455 peak, a mass 807.43 peak and a mass 813.433 peak, and a mass 829.4 peak and a mass 835.421 peak are mass pairs. It was observed as a peak pair with a number difference of 6. Further, these peak pairs were confirmed to be derived from oxidized lysozyme because the peak intensities were reversed in the mass spectrometry chart shown in FIG. 7 and the mass spectrometry chart shown in FIG.

It is a figure which shows the peak pair expressed by a label | marker. It is a figure which shows the mode of inversion of the peak intensity of a peak pair. (A) is a schematic diagram which shows ¹² C-DNPH conversion, (b) is a schematic diagram which shows ¹³ C-DNPH conversion. It is a figure which shows an example of the synthetic scheme of ¹³ C-DNPH. ^{12 is} a mass spectrometry chart of a sample obtained by enzymatic digestion of ¹² C-DNPH and ¹³ C-DNPH oxidized myoglobin, where ¹² C-DNPH protein: ¹³ C-DNPH protein = 100: 30 (molar ratio) It is a mass spectrometry chart. ^{12 is} a mass spectrometry chart of a sample obtained by enzymatic digestion of ¹² C-DNPH and ¹³ C-DNPH oxidized myoglobin, where ¹² C-DNPH protein: ¹³ C-DNPH protein = 30: 100 (molar ratio) It is a mass spectrometry chart. ^{12 is} a mass spectrometry chart of a sample obtained by enzymatic digestion of ¹² C-DNPH and ¹³ C-DNPH oxidized lysozyme, where ¹² C-DNPH protein: ¹³ C-DNPH protein = 100: 30 (molar ratio) It is a mass spectrometry chart. ^{12 is} a mass spectrometry chart of a sample obtained by enzymatic digestion of ¹² C-DNPH and ¹³ C-DNPH oxidized lysozyme, where ¹² C-DNPH protein: ¹³ C-DNPH protein = 30: 100 (molar ratio) It is a mass spectrometry chart.

Claims (7)

A method for quantifying oxidized protein, wherein the oxidized protein subjected to oxidative modification is labeled with a labeling reagent and quantified by mass spectrometry,
As the labeling reagent, a first labeling reagent that reacts with the oxidized protein, a second labeling having the same chemical structure as the first labeling reagent, and at least a part of the constituent atoms being substituted with an isotope of the atom Using the labeling reagent of
Oxidation by mixing the oxidized protein labeled with the first labeling reagent and the oxidized protein labeled with the second labeling reagent, changing the mixing ratio, and performing the mass spectrometry at each mixing ratio. A method for quantifying oxidized protein, characterized by identifying a peak derived from a protein.   The method for quantifying an oxidized protein according to claim 1, wherein the second labeling reagent has a carbon atom partially substituted with a carbon isotope.   The second labeling reagent includes a benzene ring in which six carbon atoms are substituted with carbon isotopes, and a mass difference between the first labeling reagent and the second labeling reagent is 6. Item 3. A method for quantifying an oxidized protein according to Item 2.   The method for quantifying oxidized protein according to any one of claims 1 to 3, wherein the oxidized protein is a carbonylated protein.   The first labeling reagent is 2,4-dinitrophenylhydrazine, and the second labeling reagent is 2,4-dinitrophenylhydrazine in which 6 carbon atoms of the phenyl group are substituted with carbon isotopes. The method for quantifying an oxidized protein according to claim 4,   The method for quantifying oxidized protein according to any one of claims 1 to 5, wherein the mass spectrometry is performed after enzymatic digestion of the analysis sample.   The method for quantifying an oxidized protein according to any one of claims 1 to 5, wherein the oxidized site of the oxidized protein is analyzed by tandem mass spectrometry.

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